The growth of normal cells in vitro and in vivo is regulated at a specific point in the G1 phase of the cell cycle termed the restriction point (R point). Transit through this R point is modulated by growth-regulatory hormones and factors normally supplied by the serum supplement to the growth medium. Cells that are transformed by a variety of mechanisms, such as viruses or chemicals, or that are isolated from tumors in vivo, showed a reduced requirement for serum and/or growth factors and are hypothesized to be deficient in their R point regulation. The goal of the project is the determination of the specific hormone and growth factor requirements for R point transit in several phenotypically normal cell lines and the elucidation of their mechanisms of action. It will then establish the relationships between the loss of growth factor requirements and the acquisition, following mutagenesis and selection in vitro, of a transformed, growth-unregulated phenotype. We have isolated the phenotypically normal cell lines from embryos of strains of inbred mice. They have been characterized and defined serum-free media for each are being developed. Mutants that are independent of insulin and epidermal growth factor (EGF) have been selected and are being tested for malignant phenotype. These growth-factor-independent cell lines represent possible stable intermediates in the acquisition of a transformed or malignant phenotype. We will be using them to more rigorously approach the definition of physiologic roles of oncogene products by establishing the ability or inability of known oncogenes to complement the various classes of mutants. (N)